Physical exercise is a good means of enhancing and maintaining health. However, many people struggle to make exercise be a habit, in part because of the associated fatigue. The present study investigated the effects of tomato juice on fatigue induced by exercise in humans and mice. A study of 10 healthy men and women indicated that drinking tomato juice suppressed post-exercise increases in rated perceived exertion, a subjective fatigue indicator. In mice, a single administration of tomato juice significantly attenuated exercise-induced fatigue, as measured by locomotor activity and blood transforming growth factor β and corticosterone levels. The active substances in tomatoes, which were among the aqueous components as of tomato juice supernatant, showed the same anti-fatigue effects as whole tomato juice. Moreover, these effects were mimicked by water supplemented with the aqueous components i.e., sugars, organic acids, and amino acids and were likely exerted by two or more of these components. Our results suggest that consuming tomato juice prior to or during an exercise session can reduce post-exercise fatigue, which could encourage more individuals to incorporate a regular exercise regimen into their daily routine.